Co-expression of recombinant proteins

ABSTRACT

Expression vectors are described which permit the recombinant expression of proteins which essentially contain, in addition to nucleic acid encoding the recombinant protein, nucleic acid encoding a non-proteolytic analog of Haemophilus Hin47 protein, with or without leader sequence, or nucleic acid encoding high molecular weight proteins of non-typeable Haemophilus, which are hmwB, hmwC or hmwBC.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology,biochemistry and vaccinology, in particular, to the co-expression ofrecombinant proteins.

BACKGROUND TO THE INVENTION

Recombinant proteins expressed from E. Coli, are often made as insolubleinclusion bodies. While the purification of inclusion bodies isrelatively straightforward and can lead to a >90% purification of therecombinant protein, the resulting protein is often denatured and may bebiologically inactive. In some instances, it may be advantageous tooverproduce a recombinant protein in a soluble form. Recombinantproteins can also be degraded by host proteases. Expression ofrecombinant proteins in the presence of particular proteins, such aspotential molecular chaperones, may have the effect of protecting themfrom degradation and ensure correct folding. In other instances, it maybe useful to produce two vaccine components, from different organisms,in the same production cycle. Co-expression of recombinant proteinsencoded on genes from multiple organisms can lead to improved productiontime and costs.

Otitis media is the most common illness of early childhood, with 60 to70% of all children of less than 2 years of age experiencing between oneand three ear infections (ref. 1, various references are referred to inparenthesis to more fully describe the state of the art to which thisinvention pertains. Full bibliographic information for each citation isfound at the end of the specification, immediately preceding the claims.The disclosure of these references are hereby incorporated by referenceinto the present disclosure). Chronic otitis media is responsible forhearing, speech and cognitive impairments in children. In the UnitedStates alone, treatment of otitis media costs between 1 and 2 billiondollars per year for antibiotics and surgical procedures, such astonsillectomies, adenoidectomies and the insertion of tympanostomytubes. It is estimated that an additional $30 billion is spent per annumon adjunct therapies, such as speech therapy and special educationclasses. The disease is caused by bacterial and/or viral infections, andmany of the bacteria are becoming antibiotic resistant. Infection withStreptococcus pneumoniae accounts for about 50% of bacterial disease,while non-typeable Haemophilus influenzae (NTHi) infections account forabout 30%, and Moraxella catarrhalis is responsible for about 20% ofacute otitis media. An effective prophylactic vaccine against otitismedia is thus desirable.

When under environmental stress, such as high temperature, organismsoverproduce stress response or heat shock proteins (hsps). In someinstances, hsps have also been demonstrated to be molecular chaperones(ref. 2). The bacterial HtrA or DegP heat shock proteins are expressedunder conditions of stress and the H. influenzae HtrA protein has beenshown to be a partially protective antigen in the intrabulla challengemodel of otitis media (ref. 3). The HtrA proteins are serine proteasesand their proteolytic activity makes them unstable. In addition, ascomponents of a multi-component vaccine, the wild-type HtrA proteindegrade admixed antigens. The site-directed mutagenesis of the H.influenzae htrA gene (termed hin47) and the properties of the mutantshave been fully described in U.S. Pat. No. 5,506,139 (Loosmore et al.),assigned to the assignee hereof and the disclosure of which isincorporated herein by reference. The non-proteolytic HtrA analogue,H91A Hin47, has been shown to be a protective antigen in the intrabullachinchilla model of otitis media (ref. 3). The mature H91A Hin47 proteinis produced at 40 to 50% of total E. coli protein, in a soluble form. Itmay also be produced with its leader sequence, at a level of 20 to 30%of total E. coli protein. In this form, it may function as a molecularchaperone, anchored in the periplasmic membrane (ref. 4).

During natural infection by NTHi, surface-exposed outer membraneproteins that stimulate an antibody response are potentially importanttargets for bactericidal and/or protective antibodies and are thereforepotential vaccine candidates. Barenkamp and Bodor (ref. 5) demonstratedthat convalescent sera from children suffering from otitis media due toNTHi, contained antibodies to high molecular weight (HMW) proteins.About 70 to 75% of NTHi strains express the HMW proteins, and most ofthese strains contain two gene clusters termed hmw11ABC and hmw2ABC(refs. 6, 7). The HMWA proteins have been demonstrated to be adhesinsmediating attachment to human epithelial cells (ref. 8). Immunizationwith a mixture of native HMW1A and HMW2A proteins resulted in partialprotection in the chinchilla intrabulla challenge model of otitis media(ref. 9). The production yields of native HMW proteins from H.influenzae strains are very low, but a method for producing protectiverecombinant HMW (rHMW) proteins has been described in copending U.S.patent application Ser. No. 09/167,568 filed Oct. 7, 1998 (WO 00/20609),assigned to the assignee hereof and the disclosure of which isincorporated herein by reference. The HMWB and HMWC proteins are thoughtto function as molecular chaperones, responsible for the correctprocessing and secretion of the HMWA proteins (ref. 10).

A second family of high molecular weight adhesion proteins has beenidentified in about 25% of NTHI and in encapsulated H. influenzaestrains (refs. 11, 12, 13). The NTHi member of this second family istermed Haemophilus influenzae adhesin or Hia, and the homologous proteinfound in encapsulated strains is termed Haemophilus influenzae surfacefibril protein or Hsf. The hia gene was originally cloned from anexpression library using convalescent sera from an otitis media patient,which indicates that it is an important immunogen during disease.Production of the full-length recombinant Hia protein in E. coli appearsto be toxic to the host, so a series of N-terminally truncated proteinswas made as described in copending U.S. patent application Ser. No.09/268,347 filed Mar. 16, 1999 and in PCT Patent Application No.PCT/CA00/00289 filed Mar. 16, 2000, both assigned to the assignee hereofand the disclosures of which are incorporated herein by reference. TheV38 rHia protein was chosen for further development as a vaccine, but itwas found that the first 6 amino acids of this protein were deleted froma portion of the product during synthesis in E. coli, leading to amixture of V38 rHia and S44 rHia. When an expression construct wasdeveloped to produce the S44 rHia, it was found that the N-terminus wasstable, with only S44 rHia product being made. The rHia products appearas a doublet on SDS-PAGE when expressed alone. However, whenco-expressed with H91A Hin47, the S44 rHia is produced as a single band,as described below.

The S. pneumoniae antigen, pneumococcal surface adhesin A or PsaA, is aprotective antigen in an animal model (ref 14), which is produced inhigh yield from E. coli as a 37 kDa protein. The protein may be producedas a lipoprotein if the psaA gene contains a sequence encoding alipoprotein leader sequence, or as a soluble protein if the gene encodesthe mature protein. The protein and the encoding nucleotide sequence aredescribed in U.S. Pat. No. 5,854,466, the disclosure of which isincorporated herein by reference. When co-expressed with H91A Hin47,both proteins are produced in high yield, as described below. They maybe separated by hydroxylapatite (HTP) column chromatography duringpurification, resulting in the high level production of two vaccinecomponents from different organisms, as described herein.

The over-production of E. coli chaperone proteins DnaK, DnaJ and GrpG(Hsp70) or GroEL and GroES (Hsp60) results in increased solubility ofrecombinant human protein kinases Csk, Fyn or Lck (ref. 15). Thesechaperones have also been shown to aid in the refolding of an allergen(Japanese cedar pollen) in E. coli (ref. 16). The E. coli Skp chaperonehas also been used to increase the solubility of recombinantsingle-chain antibody fragments when co-expressed in E. coli (ref. 17).All these systems use a native E. coli chaperone to aid in thesolubility and folding of recombinant proteins in E. coli. The presentinvention, for the first time, uses a heterologous protein as thechaperone

SUMMARY OF THE INVENTION

The present invention is directed to the expression of recombinantprotein and expression vectors for utilization therein. In one featureof the present invention, such expression is effected in conjunctionwith expression of non-proteolytic analogs of Haemophilus Hin47 and inanother feature of the present invention, such expression is effected inconjunction with expression of a high molecular weight protein ofnon-typeable Haemophilus which is hmwBC, hmwB or hmwC.

Accordingly, in one aspect of the present invention, there is providedan expression vector, comprising a nucleic acid molecule encoding anon-proteolytic analog of a Hin47 protein of a strain of Haemophilusincluding a portion thereof encoding the leader sequence for saidnon-proteolytic analog, and a promoter operatively connected to saidnucleic acid molecule to direct expression of said non-proteolyticanalog of a Hin47 protein having said leader sequence in a host cell.

In the various aspects of the invention involving a non-proteolyticanalog of Hin47 protein, such analog may be a mutation of natural Hin47protein in which at least one amino acid selected from the groupconsisting of amino acids 91, 121 and 195 to 201 of natural Hin47protein has been deleted or replaced by another amino acid. Preferably,the analog has histidine 91 replaced by alanine. This specific analog istermed H91A Hin47.

The vector may be a plasmid vector which may be one having theidentifying characteristics of plasmid JB-3120-2 as seen in FIG. 1A,such identifying characteristics being the nucleic acid sequences andrestriction sites identified therein.

In accordance with another aspect of the present invention, there isprovided an expression vector for expression of a recombinant protein ina host cell, comprising a nucleic acid molecule encoding anon-proteolytic analog of a Haemophilus Hin47 protein, at least oneadditional nucleic acid molecule encoding a recombinant protein, and atleast one regulatory element operatively connected to said first nucleicacid molecule and said at least one additional nucleic acid molecule toeffect expression of at least said recombinant protein in the host cell.

In one embodiment, the nucleic acid molecule encoding thenon-proteolytic analog of a Hin47 protein includes a portion thereofencoding the leader sequence of the non-proteolytic analog, or suchportion may be absent.

The at least one additional nucleic acid molecule may encode a Hia orHsf protein of a strain of Haemophilus influenzae, specifically a Hiaprotein which is N-terminally truncated. The N-terminal truncation maybe S44 or V38. The construction of such N-terminal truncations isdescribed below.

The vector may be a plasmid vector, which may be one having theidentifying characteristics of plasmid DS-2542-2-2 as seen in FIG. 5 orof plasmid JB-3145-1 seen in FIG. 10, for expression of N-terminallytruncated Hia proteins. Such identifying characteristics are the nucleicacid sequences and restriction sites seen in the respective Figures.

The at least one additional nucleic acid molecule may encode a PsaAprotein of a strain of Streptococcus pneumoniae. The vector may be aplasmid vector having the identifying characteristics of plasmidJB-3073R-1 as seen in FIG. 12 or of plasmid JB-3090-1 or JB-3090-7, asseen in FIG. 13, and expressing the PsaA protein. Such identifyingcharacteristics are the nucleic acid sequences and restriction sitesseen in the respective Figures.

The expression vector provided in this aspect of the present inventionmay be utilized in the generation of a recombinant protein by expressionfrom a suitable host cell, such as E. coli. Accordingly, in anotheraspect of the present invention, there is provided a method forexpressing at least one protein, which comprises providing a firstnucleic acid molecule encoding a non-proteolytic analog of a Hin47protein of Haemophilus;

isolating at least one additional nucleic acid molecule encoding aprotein other than Hin47; introducing the first nucleic acid moleculeand the at least one additional nucleic acid molecule into a cell toproduce a transformed cell; and growing the transformed cell to produceat least one protein. The nucleic acid molecules and regulatory elementsmay be incorporated into any of the specific expression vectorsdiscussed above.

As noted above, one feature of the present invention involves vectorsbased on nucleic acid encoding high molecular weight (HMW) protein of anon-typeable strain of Haemophilus. Accordingly, in accordance with afurther aspect of the present invention, there is provided an expressionvector, comprising a nucleic acid molecule encoding a high molecularweight protein of a non-typeable strain of Haemophilus selected from thegroup consisting of hmwB and hmwC, and a promoter operatively connectedto said nucleic acid molecule to direct expression of said highmolecular weight protein in a host cell.

The vector may be a plasmid vector which may have the identifyingcharacteristics of IN-137-1-16 shown in FIG. 18A or of pT7 hmwC shown inFIG. 19A, such identifying characteristics being the nucleic acidsequences and restrictions sites identified in the respective Figures.

The vectors, along with corresponding vectors including the hmwBC gene,may be used to construct expression vectors for the recombinantexpression of proteins in a host cell. Accordingly, a yet further aspectof the present invention provides an expression vector for expression ofa recombinant protein in a host cell, comprising a nucleic acid moleculeencoding a high molecular weight (HMW) protein of a non-typeable strainof Haemophilus selected from the group consisting of hmwBC, hmwB andhmwC, at least one additional nucleic acid molecule encoding therecombinant protein, and at least one regulatory element operativelyconnected to said first nucleic acid molecule and said at least oneadditional nucleic acid molecule to effect expression of at least saidrecombinant protein in the host cell.

In the latter vector, the at least one additional nucleic acid moleculemay be inserted into a plasmid having the identifying characteristics ofIN-52-1-13 as shown in FIG. 17A, or of IN-137-1-16 shown in FIG. 18A, orpT7 hmwC shown in FIG. 19A, under the control of the regulatoryelement(s). Such identifying characteristics are the nucleic acidmolecules and restriction sites identified in the respective Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the accompanying drawings, in which:

FIG. 1A describes the construction of vector JB-3120-2, a plasmidcontaining the H91A hin47 gene including the sequence encoding theleader sequence. Restriction sites are: B, BamH I; Bg, Bgl II; Cl, ClaI; H, Hind III; Nde, Nde I; Ps, Pst I; Pv, Pvu I; R, EcoR I; S, Sal I,Xb, Xba I; Xho, Xho I. Other abbreviations are: T7p, T7 promoter; htrA,wild-type Hin47 gene; ApR, ampicillin resistance; TetR, tetracyclineresistance; 5′f, 5′-flanking sequence; 3′f, 3′-flanking sequence. The Xmarks the site of the H91A mutation.

FIG. 1B describes the oligonucleotides used for PCR amplification of thesequence encoding the leader. Sense strand (6931.SL) SEQ ID No: 1,encoded amino acid sequence SEQ ID No: 2; anti-sense strand (6932.SL)SEQ ID No: 3, complementary strand SEQ ID No: 4, encoded amino acidsequence SEQ ID No: 5.

FIG. 2 contains an SDS-PAGE analysis of the production of H91A Hin47,with or without its leader sequence. Lane 1, H91A hin47−leader, t₀; lane2, H91A hin47−leader, t₄; lane 3, H91A hin47+leader, t₀; H91Ahin47+leader, t₄.

FIG. 3 shows the purification scheme for H91A Hin47 with leader.

FIG. 4, having Panels A and B, contains a gel analysis of the extractionof H91A Hin47 with (Panel A) or without (Panel B) its leader sequence.Lane 1, pre-stained protein molecular weight markers; lane 2, E. coliwhole cell lysates; lane 3, soluble proteins in 50 mM Tris-HCl, pH 8.0extraction; lane 4, soluble proteins in 50 mM Tris-HCl, pH 8.0/0.5%Triton X-100/10 mM EDTA extraction; lane 5, pellets after the twoextractions.

FIG. 5 shows the construction of vector DS-2342-2-2, a plasmidcontaining the T7 H91A hin47 and T7 V38 hia gene cassettes. The H91Ahin47 gene encodes the mature protein. Restriction sites are: B, BamH I;Bg, Bgl II; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I; S, Sal I.Other abbreviations are: CAP, calf alkaline phosphatase; T7p, T7promoter; ApR, ampicillin resistance; KanR, kanamycin resistance; TetR,tetracycline resistance.

FIG. 6 contains an SDS-PAGE analysis of the production of V38 rHia andH91A Hin47, when co-expressed from the same plasmid. Lane 1, V38rHia+H91A Hin47, t₀; Lane 2, V38 rHia+H91A Hin47, t4; V38 rHia, t₄.

FIG. 7 shows the purification scheme for V38 rHia when co-expressed withH91A Hin47.

FIG. 8 contains an SDS-PAGE analysis of the purification of V38 rHiaafter co-expression with H91A Hin47. Lane 1, Prestained molecular weightmarkers; lane 2, E. coli cell lysate; lane 3, soluble proteins after 50mM Tris-HCl, pH 8.0/0.1 M NaCl extraction; lane 4, soluble proteins in50 mM Tris-HCl. pH 8.0/0.5% Triton X-100/10 mM EDTA extraction; lane 5,soluble proteins in 50 mM Tris-HCl, pH 8.0/1% octylglucoside extraction;lane 6, flow-through fraction after DEAE-Sephacel column; lane 7,flow-through fraction after HTP column; lane 8, purified H91A Hin47;lane 9, purified rHia protein.

FIG. 9 shows the construction of vector JB-3134-1-1, a plasmidcontaining the T7 H91A hin47 and T7 S44 hia gene cassettes. The H91Ahin47 gene encodes the mature protein. Restriction sites are: B, BamH I;Bg, Bgl II; Cl, Cla I; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I.Other abbreviations are: CAP, calf alkaline phosphatase; T7p, T7promoter; ApR, ampicillin resistance; KanR, kanamycin resistance.

FIG. 10 shows the construction of vector JB-3145-1, a plasmid containingthe T7 H91A hin47 and T7 S44 hia gene cassettes. The H91A hin47 geneencodes the protein with its leader sequence. Restriction sites are: B,BamH I; Bg, Bgl II; Cl, Cla I; H, Hind III; Nde, Nde I; Ps, Pst I; R,EcoR I. Other abbreviations are: CAP, calf alkaline phosphatase; T7p, T7promoter; ApR, ampicillin resistance; KanR, kanamycin resistance.

FIG. 11 contains an SDS-PAGE analysis of the production of S44 rHia andH91A Hin47±leader, when co-expressed from the same plasmid. Lane 1, H91AHin47−leader, t₀; lane 2, H91A Hin47−leader, t₄; lane 3, H91AHin47+leader, t₄; lane 4, S44 rHia, t₄; lane 5, H91A Hin47 (−L)+S44rHia, t₄; lane 6, H91A Hin47 (+L)+S44 rHia, t₄.

FIG. 12A shows the construction of vector JB-3073R-1, a plasmidcontaining the T7 psaA and T7 H91A hin47 gene cassettes. The psaA geneencodes its endogenous leader sequence. Restriction sites are: B, BamHI; Bg, Bgl II; Cl, Cla I; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I,Xb, Xba I. Other abbreviations are: CAP, calf alkaline phosphatase; T7p,T7 promoter; ApR, ampicillin resistance; KanR*, kanamycin resistancegene with internal Hind III and Xho I sites deleted.

FIG. 12B shows the oligonucleotide primers used to amplify psaA (+leader). Sense strand (6850.SL), SEQ ID No: 26, encoded amino acidsequence, SEQ ID No: 27; anti-sense strand (6852.SL) SEQ ID No: 28,complementary strand, SEQ ID No: 29.

FIG. 13A shows the construction of vectors JB-3090-1 and JB-3090-7,plasmids containing the T7 psaA and T7 H91A hin47 gene cassettes, indifferent orientations. The psaA gene encodes the mature protein.Restriction sites are: B, BamHI; Bg, Bgl II; Cl, Cla I; H, Hind III;Nde, Nde I; Ps, Pst I; R, EcoR I, Xb, Xba I. Other abbreviations are:CAP, calf alkaline phosphatase; T7p, T7 promoter; ApR, ampicillinresistance; KanR*, kanamycin resistance gene with internal Hind III andXho I sites deleted.

FIG. 13B shows the oligonucleotide primers used to amplify psaA (−leader). Sense strand (6851.SL), SEQ ID No: 30, encoded amino acidsequence, SEQ ID No: 31; anti-sense strand (6852.SL) SEQ ID No: 28,complementary strand, SEQ ID No: 29.

FIG. 14 contains an SDS-PAGE analysis of the production of rPsaA±leaderand H91A Hin47 proteins, when co-expressed from the same plasmid. Lane1, H91A Hin47+rPsaA (+L) at to; lane 2, H91A Hin47+rPsaA (+L) at t₄ (<>orientation); lane 3, H91A Hin47+rPsaA (+L) at t₄ (>> orientation); lane4, H91A Hin47+rPsaA (−L) at t₄ (>> orientation).

FIG. 15 shows the purification scheme for H91A Hin47 and rPsaA (withoutleader), when co-produced.

FIG. 16, having Panels A and B, contains SDS-PAGE analysis of thepurification of H91A Hin47 (Panel A) and rPsaA (Panel B) (withoutleader). Lane 1, Prestained molecular weight markers; lane 2, E. colicell lysate; lane 3, soluble proteins after 50 mM Tris-HCl, pH 8.0extraction; lane 4, purification on DEAE-Sephacel column; lane 5,purification on HTP column; lane 6, flow-through fraction afterSartobind Q membrane, purified H91A Hin47 protein or rPsaA.

FIG. 17A describes the construction of IN-52-1-13 that co-expresses theH. influenzae hmwB and hmwC genes. Restriction sites are: B, BamH I; Bg,Bgl II; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I; S, Sal I; Xba,Xba I; Xho, Xho I. Other abbreviations are: T7p, T7 promoter; ApR,ampicillin resistance; KanR, kanamycin resistance.

FIG. 17B illustrates the oligonucleotide primers used to PCR amplify theNde I-EcoR I 5′ hmwB fragment. Sense strand (7072.SL) SEQ ID No: 6,encoded amino acid sequence SEQ ID No: 7; anti-sense strand (5950.SL)SEQ ID No: 8, complementary strand SEQ ID No: 9, encoded amino acidsequence SEQ ID No: 10.

FIG. 18A shows the construction of IN-137-1-16 to express the H.influenzae hmwB gene alone. Restriction sites are: B, BamH I; Bg, BglII; Cl, Cla I; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I, Xho, XhoI. Other abbreviations are: CAP, calf alkaline phosphatase; T7p, T7promoter; ApR, ampicillin resistance.

FIG. 18B illustrates the oligonucleotides used for construction of theHind III-BamHI 3′ hmwB fragment. 7073.SL, SEQ ID No: 11; 7074.SL, SEQ IDNo: 12; encoded amino acid sequence, SEQ ID No: 13; 7075.SL, SEQ ID No:14, 7076.SL, SEQ ID No: 15.

FIG. 19A shows the construction of a plasmid to express the H.influenzae hmwC gene. Restriction sites are: B, BamH I; Bg, Bgl II; Cl,Cla I; H, Hind III; Nde, Nde I; Ps, Pst I; R, EcoR I; Xho, Xho I. Otherabbreviations are: CAP, calf alkaline phosphatase; T7p, T7 promoter;ApR, ampicillin resistance.

FIG. 19B illustrates the oligonucleotide primers used to PCR amplify theNde I-Xho I 5′ hmwC fragment. Sense strand (7077.SL) SEQ ID No: 16,encoded amino acid sequence, SEQ ID No: 17; anti-sense strand (7078.SL)SEQ ID No: 18, complementary strand SEQ ID No: 19, encoded amino acidsequence SEQ ID No: 20.

FIG. 19C illustrates the oligonucleotides used to construct the HindIII-BamH I 3′ hmwC fragment. 7079.SL, SEQ ID No.: 21; 7080.SL, SEQ IDNo: 22; encoded amino acid sequence, SEQ ID No: 23; 7081.SL, SEQ ID No:24; 7082.SL, SEQ ID No: 25.

GENERAL DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following sections:

1. Production of Recombinant H. influenzae H91A Hin47 Protein with itsLeader Sequence.

The native bacterial HtrA protein (H. influenzae Hin47) is a stressresponse protein located in the periplasmic membrane and responsible forsurvival of the organism under stress conditions, such as hightemperature. It is a serine protease that degrades improperly folded denovo synthesized proteins. In the aforementioned U.S. Pat. No.5,506,139, there is described the production of high yield (40 to 50% oftotal E. coli proteins), soluble, mature wild-type recombinant H.influenzae Hin47 protein from E. coli. The rHtrA (rHin47) protein hadserine protease activity, which rendered it unstable after purification.Several analogues of Hin47 were generated by site-directed mutagenesisand the H91A Hin47 recombinant protein was found to be stable, of highyield, and protective in animal models. It had lost all measurableserine protease activity. When produced as the soluble mature protein,H91A Hin47 seemed to increase the solubility of co-produced proteins, aproperty which can be advantageous, as described below.

Stress response proteins may function as chaperones, serving tostabilize other expressed proteins. The H91A Hin47 protein has beenproduced with its endogenous leader sequence in an attempt to localizeit to the periplasmic membrane and mimic the HtrA chaperone function.Since the endogenous serine protease activity has been ablated, it washoped that H91A Hin47 might stabilize co-produced proteins in E. coli.The H91A Hin47 (+ leader) protein was made at 20 to 25% of total E. coliprotein (FIG. 2) and was found to be insoluble after extraction withTriton X-100, suggesting that it was expressed either as amembrane-bound protein or inclusion bodies (FIG. 4).

2. Production of Recombinant H. influenzae Hia Protein in the Presenceof H91A Hin47, with or without a Leader Sequence.

The H. influenzae Hia or Hsf proteins are demonstrated adhesins and assuch are important vaccine candidates. The production of recombinant H.influenzae Hia proteins from E. coli has been described in theaforementioned U.S. patent application Ser. No. 09/268,347. Thefull-length proteins were expressed at very low levels and wereapparently toxic to E. coli. A series of truncated rHia proteins wasmade, which were sequentially deleted at the N-terminus. The V38 rHiaprotein was produced as “soft” inclusion bodies and was purified, asdescribed in the aforementioned U.S. patent application Ser. No.09/268,347. When the V38 rHia protein was co-produced with mature H91AHin47, its solubility was increased. This led to an improved recoveryduring protein purification, and represents a novel use of mature H91AHin47 (FIG. 8). When analysed by SDS-PAGE, the V38 rHia protein wasapparently produced as two doublets, whether or not it was co-producedwith mature H91A Hin47 (FIG. 6).

The S44 rHia protein, prepared as described in the aforementioned PCTPatent Application No. PCT/CA00/00289 filed Mar. 16, 2000, was alsoproduced as “soft” inclusion bodies and was purified by the same processas the V38 rHia protein. When analysed by SDS-PAGE, the S44 rHia proteinwas apparently produced as two doublets, if produced alone. When S44rHia was co-produced with mature H91A Hin47, SDS-PAGE analysis revealedthat it was apparently a single species (FIG. 11). This apparentstabilization of a co-produced protein represents a novel use of matureH91A Hin47.

3. Production of Recombinant S. pneumoniae PsaA Protein in the Presenceof H. influenzae H91A Hin47.

The majority of acute bacterial otitis media is caused by S. pneumoniae,H. influenzae and M. catarrhalis infections. A broadly effective vaccineagainst this disease would ideally include antigens from all threeorganisms. The production of a multi-component vaccine based uponrecombinant proteins can be time-consuming and/or costly. If antigenscould be co-produced, the cycle time for vaccine preparation could bereduced. In order for this to be effective, the antigens should be madein similar quantities, if they are to be combined in a 1:1 ratio in thefinal vaccine. It must also be possible to separate them duringpurification.

The S. pneumoniae PsaA protein is a demonstrated adhesin that isprotective in an animal model, and, as such, represents an importantvaccine candidate. The native PsaA protein is a lipoprotein. Therecombinant mature PsaA and lipo PsaA proteins are both made in highyield (30 to 40% of total protein) from E. coli. The mature protein isproduced as a soluble protein and the lipoprotein appears to bemembrane-associated. The recombinant mature H91A Hin47 vaccine componentis also produced in high yield at 40 to 50% of total E. coli proteins.When the rPsaA and H91A Hin47 proteins are co-produced, they are stillmade in high yield, at 20 to 30% of total protein each (FIG. 14).

The procedure for purification of the mature rPsaA and H91A Hin47proteins is shown in FIG. 16. Both H91A Hin47 and rPsaA (without leader)were expressed as soluble proteins. Separation of H91A Hin47 from rPsaAwas achieved on a DEAE-Sephacel column, to which rPsaA bound, whereasH91A Hin47 did not. Further purification of both proteins included HTPchromatography and Sartobind Q-membrane.

4. Production of Recombinant H. influenzae HMWB and/or HMWC Proteins asPotential Chaperones.

The H. influenzae HMWA protein is a demonstrated adhesin that isprotective in animal models. The production of rHMWA proteins has beendescribed in the aforementioned U.S. patent application Ser. No.09/167,568. The H. influenzae HMWA protein is produced as a largeprecursor, from which a 35 kDa N-terminal fragment is cleaved duringprocessing and secretion. The H. influenzae HMWA protein is encoded aspart of an operon, hmwABC, that also encodes two accessory proteinstermed HMWB and HMWC, that are thought to function as chaperones. TherHMWB and rHMWC proteins are made in good yield from E. coli, whenexpressed from the hmwABC operon. It has been demonstrated that theproperties of a recombinant protein can be significantly altered whenco-produced with the putative chaperone H91A Hin47. It would beinteresting to determine what effect there would be on recombinantproteins co-produced with the H. influenzae rHMWB and/or rHMWC putativechaperone proteins.

It would be advantageous to express the hmwB and hmwC genes separately.Therefore, vectors have been designed to express the individual hmwB,hmwC, or hmwBC genes. Other genes encoding proteins of interest may beco-expressed with the hmwB, hmwC, or hmwBC genes.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, immunology andfermentation technology used, but not explicitly described in thisdisclosure and these Examples, are amply reported in the scientificliterature and are well within the ability of those skilled in the art.

Example 1

This Example describes the construction of plasmid JB-3120-2, whichcontains the T7 H91A hin47 gene encoding the endogenous leader sequence.The procedure employed is shown schematically in FIG. 1A.

The production of the mature recombinant H91A Hin47 protein from E. colihas been described in the aforementioned U.S. Pat. No. 5,506,139. Thisprotein was produced at 40 to 50% of total protein in a soluble form.The bacterial HtrA proteins are located in the periplasmic membrane andmay function as chaperones if located there. In order to direct themutant, non-proteolytic H91A Hin47 protein to the periplasmic membrane,the endogenous leader was added. Plasmid DS-2140-3 is a pBR328-basedplasmid that contains the T7 H91A hin47 gene cassette between EcoR I andCla I sites (FIG. 1A). Plasmid JB-1 172-2-5 is a pUC-based plasmid thatcontains the wild-type htrA gene with 5′- and 3′-flanking sequences.Plasmid JB-1172-2-5 was digested with Cla I and Sal I and the 0.6 kb3′-flanking fragment was purified. Plasmid DS-2140-3 was digested withCla I and Sal I, the 5.1 kb fragment purified, and the 3′-flankingfragment inserted, to generate plasmid JB-2706-9. Plasmid DS-1843-2 is apBR328-based vector into which a multiple cloning site has beeninserted. JB-2706-9 was digested with EcoR I and Sal I, releasing the2.5 kb T7 H91A hin47/3+f gene sequence. The EcoR I-Sal I fragment wasinserted into DS-1843-2, that had been digested with EcoR I and Xho I,generating plasmid JB-2721-1. Plasmid JB-1172-2-5 was digested withEcoRI and Pvu I to release the 0.6 kb 5′-flanking sequence. PlasmidJB-2721-1 was digested with EcoR I and Pvu I to delete the T7 promotersequence and the 5′-flanking sequence was inserted, generating plasmidJB-2750-8, that contains a genomic 5′-flanking/htrA*/3′-flankingsequence with the H91A mutation. The HtrA leader sequence was PCRamplified from JB-2750-8 on a 0.25 kb Nde I-Pvu I fragment, using theoligonucleotide primers shown in FIG. 1B. Plasmid JB-2750-8 was digestedwith Pvu I and Pst I and the 1.7 kb H91A hin47/3′-flanking fragment waspurified. Vector pT7-7 was digested with Nde I and Pst I and the NdeI-Pvu I and Pvu I-Pst I fragments inserted, to generate plasmidJB-3120-2. Plasmid DNA was introduced into electrocompetent E. coliBL21(DE3) cells using a BioRad electroporator and recombinant E. colistrain JB-3129-1 was grown for protein analysis, as described in thefollowing Example.

Example 2

This Example describes the production and purification of recombinantH91A Hin47 protein with its endogenous leader sequence.

Cells were grown at 37° C. in NZCYM medium using the appropriateantibiotic selection to A₅₇₈ of 0.3 before addition of lactose to 1.0%for 4 hours. Samples were adjusted to 0.2 OD/μl with SDS-PAGElysis+loading buffer and the same amount of each protein sample wasloaded onto SDS-PAGE gels (ref 18). The mature H91A Hin47 protein wasproduced at ˜50% of total protein, while the H91A Hin47+leader proteinwas produced at 20 to 25% of total protein (FIG. 2).

The purification of the mature soluble H91A Hin47 protein has beendescribed in U.S. Pat. No. 5,506,139. The H91A Hin47 +leader protein wasfound to be associated with the pellet after two extractions of E. colicells with 50 mM Tris-HCl, pH 8.0 and 50 mM Tris-HCl, pH 8.0 containing0.5% Triton X-100 and 10 mM EDTA. The pellet containing H91A Hin47 wassolubilized in 50 mM Tris-HCl, pH 8.0, containing 8M urea. After thepellets were dissolved, 50 mM Tris-HCl, pH 8.0 was added to bring thefinal urea concentration to 2 M.

The above solution was then applied to a Macro-prep ceramichydroxyapatite column (HTP, Bio-Rad Laboratories) equilibrated in 10 mMNa—PO₄ buffer, pH 8.0. H91A Hin47 protein bound to the HTP column. Afterwashing the column with 10 column volumes of 175 mM Na—PO₄, pH 8.0, H91AHin47 was eluted from the HTP with 0.3 M Na—PO4, pH 8.0. The amount ofH91A Hin47 in the elution fractions was determined by the bicinchoninicacid (BCA) protein assay using BSA as a standard. The purity of finalpreparation was assessed by SDS-PAGE analysis.

Example 3

This Example illustrates the construction of plasmid DS-2342-2-2, whichcontains the T7 H91A hin47, T7 V38 hia, and E. coli cer genes. Theprocedure employed is shown schematically in FIG. 5.

Plasmid DS-1872-2-2 is a pBR328-based vector containing a 2.2 kb EcoR IT7 H91A hin47 gene cassette (FIG. 5). Plasmid BK-96-2-11 is apBR328-based vector that contains a T7 V38 hia gene cassette, the E.coli cer gene, and a kanamycin resistance gene; and this plasmid hasbeen described in the aforementioned U.S. patent application Ser. No.09/268,347. BK-96-2-11 was linearized by digestion with EcoR I,dephosphorylated, and the EcoR I T7 H91A hin47 gene fragment inserted,to generate plasmid DS-2342-2-2, that co-expresses the H91A hin47 andV38 hia genes. This plasmid thus contains tandem T7 H91A hin47 and T7V38 hia genes in the same orientation. Plasmid DNA was introduced intoelectrocompetent E. coli BL21(DE3) cells using a BioRad electroporator,and recombinant E. coli strain DS-2350-3-1 was grown for proteinanalysis, as described in the following Example.

Example 4

This Example describes the production and purification of recombinantV38 rHia protein that was co-produced with H91A Hin47.

Protein samples were prepared and analysed as described in Example 2.The V38 rHia and mature H91A Hin47 proteins were both produced uponinduction (FIG. 6). The V38 rHia protein appeared as a pair of doubletson SDS-PAGE, whether or not it was produced in the presence of H91AHin47.

The purification of V38 rHia, produced as inclusion bodies, has beendescribed in U.S. patent application Ser. No. 09/268,347. Whenco-produced with H91A Hin47, the V38 rHia protein was apparently moresoluble; and the majority of rHia protein was recovered in the initial50 mM Tris-HCl, pH 8.0/0.1 M NaCl extraction. As shown in FIGS. 7 and 8,the separation of rHia from H91A Hin47 was achieved through a HTPcolumn, to which H91A Hin47 protein bound but rHia did not. Afterconcentration of rHia by PEG 4000 or ammonium sulfate, the protein wasfurther purified on a Superdex 200 gel filtration column, the sameprocess used for the purification of rHia expressed as inclusion bodies.A Sartibond Q membrane was used as a final polishing step to furtherremove LPS and residual contaminants. The purity of rHia or H91A Hin47was assessed by SDS-PAGE analysis (FIG. 8), according to the procedureof Laemmli (ref. 18).

Example 5

This Example illustrates the construction of plasmid JB-3134-1-1, whichcontains the T7 S44 hia and T7 H9]A hin47 (no leader) genes. Theprocedure employed is shown schematically in FIG. 9.

Plasmid DS-1298-1 is a pBR322-based plasmid (pEV, ref 19) that containsthe T7 H91A hin47 gene on a 2.2 kb Bgl II-BamH I fragment (FIG. 9).Plasmid JB-2930-3 is a pBR328-based vector that contains the T7 S44 hia,E. coli cer, and kanamycin resistance genes and is described in theaforementioned PCT Patent Application No. PCT/CA00/00289. PlasmidJB-2930-3 was linearized by digestion with Bgl II, dephosphorylated, andthe Bgl II-BamH I T7 H91A hin47 gene fragment inserted to generateplasmid JB-3134-1-1. This plasmid thus contains tandem T7 H91A hin47(−L) and T7 S44 hia genes in the same orientation. Plasmid DNA wasintroduced into electrocompetent E. coli BL21(DE3) cells using a BioRadelectroporator, and recombinant E. coli strain JB-3144-1 was grown forprotein analysis.

Example 6

This Example illustrates the construction of plasmid JB-3145-1, whichcontains the T7 S44 hia and T7 H91A hin47 (with leader) genes. Theprocedure employed is shown schematically in FIG. 10.

Plasmid JB-3120-2, prepared as described in Example 1, contains the T7H91A hin47 (+ leader) cassette on a Bgl II-Cla I fragment (FIG. 10).Plasmid pEV vrf2 is a pBR322-based plasmid containing the γP_(L)promoter and a multiple cloning site (ref. 19). Plasmid pEV vrf2 wasdigested with Bgl II and Cla I, and the T7 H91A hin47 (+L) gene cassettewas inserted to generate plasmid JB-3133-1-1. This plasmid contains theT7 H91A hin47 (+L) gene cassette on a Bgl II-BamH I fragment. PlasmidJB-2930-3 is a pBR328-based vector that contains the T7 S44 hia, E. colicer, and kanamycin resistance genes, and is prepared as described in theaforementioned PCT Patent Application No. PCT/CA00/00289. PlasmidJB-2930-3 was digested with Bgl II, dephosphorylated, and the BglII-BamH I T7 H9]A hin47 (+L) fragment was inserted to generate plasmidJB-3145-1. This plasmid thus contains tandem T7 H91A hin47 (+L) and T7S44 hia genes in the same orientation. Plasmid DNA was introduced intoelectrocompetent E. coli BL21(DE3) cells using a BioRad electroporatorand recombinant E. coli strain JB-3153-1-1 was grown for proteinanalysis.

Example 7

This Example describes the production of recombinant S44 rHia proteinthat was co-produced with H91A Hin47±leader.

Protein samples, produced by the plasmids JB-3134-1-1 and JB-3145-1,described in Examples 5 and 6, were prepared and analysed as describedin Example 2. The S44 rHia and mature H91A Hin47 proteins were bothproduced upon induction (FIG. 11). The S44 rHia protein appeared as apair of doublets when expressed alone, but as a single band whenco-expressed with H91A Hin47 (−L). The H91A Hin47 protein appears tohave enhanced the stability of the co-produced S44 rHia protein. The S44rHia and H91A Hin47 (with leader) proteins were both produced uponinduction, although the amount of S44 rHia was significantly reduced(FIG. 11).

Example 8

This Example describes the construction of plasmid JB-3073R-1, whichcontains the T7 H91A hin47 and T7 psaA (with leader) genes. Theprocedure employed is shown schematically in FIG. 12A.

The H. influenzae H91A Hin47 and S. pneumoniae rPsaA proteins are bothpotential vaccine candidates and are made in high yield from E. coliwhen expressed individually. The production time for these vaccineantigens can be significantly reduced if they could be co-expressed andseparated by purification. Plasmid JB-2996-1-6 is an ampicillinresistant pET-based vector containing a T7 psaA (+ leader) gene cassetteencoding the 37 kDa lipo rPsaA protein (FIG. 12A). PCR primers foramplification of the psaA (+ leader) gene are described in FIG. 12B.Plasmid JB-3004-26 was derived from plasmid pUC-4K (Pharmacia) bysite-directed mutagenesis of the kanamycin resistance (kanR) gene. Theinterior Hind HI and Xho I sites were deleted, but the Cla I and Sma Isites were unchanged. Plasmid JB-2996-1-6 was linearized with Pst I,dephosphorylated, and the mutated kanR gene from JB-3004-26 was insertedto generate JB-3060-1-25. Plasmid DS-1298-1 is a pBR322-based plasmidcontaining the T7 H91A hin47 gene, encoding the mature H91A Hin47protein, on a 2.2 kb Bgl H-BamH I fragment. Plasmid JB-3060-1-25 waslinearized with Bgl II, dephosphorylated, and the Bgl II-BamH I T7 H91Ahin47 gene inserted to generate JB-3073R-1. This plasmid thus containstandem T7 H91A hin47 and T7psaA (+L) genes in the same orientation.Plasmid DNA was introduced into electrocompetent E. coli BL21(DE3) cellsusing a BioRad electroporator, and recombinant E. coli strain IA-181-1was grown for protein analysis.

Example 9

This Example describes the construction of plasmids JB-3090-1 andJB-3090-7, which contain the T7 H91A hin47 and T7 psaA (no leader)genes. The procedure employed is shown schematically in FIG. 13A.

Plasmid JB-2996-2-2 is an ampicillin resistant pET-based vectorcontaining a T7 psaA (− leader) gene cassette, encoding the mature rPsaAprotein (FIG. 13A). PCR primers to amplify psaA (− leader) gene aredescribed in FIG. 13B. Plasmid JB-3004-26 contains the mutated kanamycinresistance gene, as described in Example 8. Plasmid JB-2996-2-2 waslinearized with Pst I, dephosphorylated, and the mutated kanR gene fromJB-3004-26 was inserted to generate JB-3060-2-5. Plasmid DS-1298-1 is apBR322-based plasmid containing the T7 H91A hin47 gene encoding themature H91A Hin47 protein on a 2.2 kb Bgl II-BamH I fragment. PlasmidJB-3060-2-5 was linearized with Bgl II, dephosphorylated, and the BglII-BamH I T7 H91A hin47 gene inserted to generate plasmids JB-3090-1 andJB-3090-7. These plasmids differ only in the relative orientation of theinserted T7 H91A hin47 gene. Plasmid JB-3090-7 thus contains tandem T7H91A hin47 and T7psaA (−L) genes in the same orientation, while plasmidJB-3090-1 contains tandem T7 H91A hin47 and T7psaA (−L) genes inopposite orientations. It has been noted that the latter arrangement ofgenes is very rarely cloned. Plasmid DNA was introduced intoelectrocompetent E. coli BL21(DE3) cells using a BioRad electroporator;and recombinant E. coli strains JB-3106-1-1 (from JB-3090-1) andJB-3106-2-1 (from JB-3090-7) were grown for protein analysis.

Example 10

This Example describes the production and purification of recombinantH91A Hin47 and PsaA±leader when co-produced from the same plasmid.

Protein samples, produced by plasmids JB-3073R-1, plasmid JB-3090-1 andplasmid JB-3090-7, described in Examples 8 and 9, were prepared andanalysed as described in Example 2. The rPsaA protein was produced ingood yield from all strains, with or without its leader sequence (FIG.14). However, the H91A Hin47 protein was produced from only two of threestrains. When the tandem genes were arranged in the same orientation,both rPsaA and H91A Hin47 were produced in high yield. StrainJB-3106-1-1, generated from plasmid JB-3090-1 that contains the tandemgenes in opposite orientations, did not produce any H91A Hin47.

The scheme for separation and purification of the H91A Hin47 and rPsaAproteins is shown in FIG. 15. Both H91A Hin47 and rPsaA (without leader)were expressed as soluble proteins. After extraction with 50 mMTris-HCl, pH 8.0, the soluble sonicate fraction was applied to aDEAE-Sephacel column equilibrated in 50 mM Tris-HCl, pH 8.0. Themajority of H91A Hin47 did not bind to the column and was recovered inthe flow-through fraction. In contrast, the majority of rPsaA bound tothe DEAE-Sephacel. After washing with 10 column volumes of 50 mMTris-HCl;, pH 8.0/10 mM NaCl to remove contaminants, rPsaA was eluted in50 mM Tris-HCl, pH 8.0 containing 30 mM NaCl. The H91A Hin47 or rPsaAfraction was further purified separately onto a Macro-prep ceramichydroxylapatite column (HTP) equilibrated in 10 mM Na—PO4 buffer, pH8.0. Both proteins bound to the HTP column. For the purification of H91AHin47, the HTP column was washed with 10 column volumes of 175 mMNa—PO4, pH 8.0, and H91A Hin47 was eluted with 0.3 M Na—PO4, pH 8.0. Forthe purification of rPsaA, the HTP column was washed with 10 columnvolumes of 50 mM Na—PO4, pH 8.0, and rPsaA was eluted with 0.2 M Na—PO4,pH 8.0. The purity of H91A Hin47 or rPsaA was assessed by SDS-PAGEanalysis (FIG. 16).

Example 11

This Example describes the construction of plasmid IN-52-1-13 thatco-expresses H. influenzae hmwB and hmwC genes. The procedure employedis shown schematically in FIG. 17A.

Plasmid JB-2641-1 is a pT7-based plasmid that contains the hmwABC geneswith an Xba I site inserted at the 3 ′-end of hmwA (FIG. 17A) and hasbeen described in the aforementioned U.S. patent application Ser. No.09/167,568. Digestion with Nde I and EcoR I deletes a 610 bp fragmentcontaining the hmwA gene and the 5′-end of the hmwB gene. The 5′-end ofhmwB is created by PCR amplification that also introduces an Nde I siteencoding a start Met, using the oligonucleotide primers shown in FIG.17B. The 460 bp Nde I-EcoR I PCR fragment is inserted into the NdeI-EcoR I vector to generate pT7 hmwBC/cer/kanR (IN47-1). In order tointroduce additional restriction enzyme sites for future constructions,the Nde I-Sal I fragment containing the complete T7 hmwBC gene cassetteis inserted into pT7-7 to generate pT7 hmwBC (IN-52-1-13).

Example 12

This Example describes the construction of plasmid IN-137-1-16 thatexpresses H. influenzae hmwB alone. The procedure employed is shownschematically in FIG. 18A.

The construction of plasmid pT7 hmwBC (IN-52-1-13) is described inExample 11. Digestion of plasmid IN-52-1-13 with Bgl II and Hind IIIgenerates a fragment containing the T7 promoter and most of the hmwBgene (FIG. 18A). The 3′-end of hmwB is synthesized as a ˜123 bp HindIII-BamH I fragment, using the oligonucleotides shown in FIG. 18B. TheBgl II-Hind III and Hind III-BamH I fragments are inserted into pT7-7that has been digested with Bgl II and BamH I, then dephosphorylated.The resultant plasmid, pT7 hmwB (IN-137-1-16), contains the T7 promoterand the full-length hmwB gene only, as a Bgl II-BamH I cassette that canbe used for co-expression studies.

Example 13

This Example describes the construction of a plasmid to express H.influenzae hmwC alone. The procedure employed is shown schematically inFIG. 19A.

The construction of plasmid pT7 hmwBC (IN-52-1-13) was described inExample 11. This vector contains the complete hmwB and hmwC genes, aswell as ˜1.3 kb of 3′-flanking sequence. Digestion of IN-52-1-13 withNde I and Xho I deletes the hmwB gene and the 5′-end of hmwC gene(FIG.19A). The 5′-end of hmwC is created by PCR amplification that alsointroduces an Nde I site encoding a start Met. The oligonucleotideprimers employed are shown in FIG. 19B. The 950 bp Nde I-Xho I 5′ hmwCPCR fragment is inserted into the Nde I-Xho I vector to generate pT7hmwC/3′f(IN-109-1). In order to create a plasmid containing only thehmwC gene with no 3′-flanking sequence, IN-109-1 was digested with BglII and Hind III and the ˜2.2 kb fragment containing the T7 promoter andmost of the hmwC gene was purified. The 3′-end of the hmwC gene issynthesized as a 80 bp Hind III-BamH I fragment from theoligonucleotides shown in FIG. 19C. The Bgl II-Hind III and HindIII-BamH I fragments are inserted into pT7-7 that has been digested withBgl II and BamH I and dephosphorylated. The resultant plasmid, pT7 hmwC,contains the T7 promoter and the full-length hmwC gene only as a BglII-BamH I cassette that can be used for co-expression studies.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, there is provided a heterologouschaperone effect in the expression of recombinant proteins.Modifications are possible within the scope of the invention.

REFERENCES

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1. An expression vector, comprising: a nucleic acid molecule encoding anon-proteolytic analog of a Hin47 protein of a strain of Haemophilusincluding a portion thereof encoding the leader sequence for saidnon-proteolytic analog, and a promoter operatively connected to saidnucleic acid molecule to direct expression of said non-proteolyticanalog of a Hin47 protein having said leader sequence in a host cell. 2.The vector of claim 1 wherein said non-proteolytic analog of Hin47protein is a mutation of natural Hin47 protein in which at least oneamino acid selected from the group consisting of amino acids 91, 121 and195 to 201 of natural Hin47 protein has been deleted or replaced by adifferent amino acid.
 3. The vector of claim 2 wherein histidine 91 isreplaced by alanine.
 4. The vector of claim 1 which is plasmid vectorhaving the identifying characteristics of plasmid JB-3120-2 as seen inFIG. 1A.
 5. An expression vector for expression of a recombinant proteinin a host cell, comprising: a nucleic acid molecule encoding anon-proteolytic analog of a Haemophilus Hin47 protein, at least oneadditional nucleic acid molecule encoding the recombinant protein, andat least one regulatory element operatively connected to said firstnucleic acid molecule and said at least one additional nucleic acidmolecule to effect expression of at least said recombinant protein inthe host cell.
 6. The vector of claim 5 wherein said nucleic acidmolecule encoding the non-proteolytic analog of a Hin47 protein includesa portion encoding the leader sequence for said non-proteolytic analog.7. The vector of claim 6 wherein said non-proteolytic analog of Hin47protein is a mutation of natural Hin47 protein in which at least oneamino acid selected from the group consisting of amino acids 91, 121 and195 to 201 of natural Hin47 protein has been deleted or replaced by adifferent amino acid.
 8. The vector of claim 7 wherein histidine 91 isreplaced by alanine.
 9. The vector of claim 5 wherein said at least oneadditional nucleic acid molecule encodes a Hia or Hsf protein of astrain of Haemophilus influenzae.
 10. The vector of claim 9 wherein saidat least one additional nucleic acid molecule encodes a Hia proteinwhich is N-terminally truncated.
 11. The vector of claim 10 wherein saidN-terminal truncation is S44 or V3
 8. 12. The vector of claim 11 whichis a plasmid vector having the identifying characteristics of plasmidDS-2342-2-2 as seen in FIG.
 5. 13. The vector of claim 11 which is aplasmid vector having the identifying characteristics of plasmidJB-3145-1 seen in FIG.
 10. 14. The vector of claim 5 wherein said atleast one additional nucleic acid molecule encodes a PsaA protein of astrain of Streptococcus pneumoniae.
 15. The vector of claim 14 which isa plasmid vector having the identifying characteristics of plasmidJB-3073R-1 as seen in FIG.
 12. 16. The vector of claim 14 which is aplasmid vector having the identifying characteristics of plasmidJB-3090-1 or JB-3090-7 as seen in FIG.
 13. 17. A method for expressingat least one protein, which comprises: providing a first nucleic acidmolecule encoding a non-proteolytic analog of a Hin47 protein ofHaemophilus; isolating at least one additional nucleic acid moleculeencoding a protein other than Hin47; introducing the first nucleic acidmolecule and the at least one additional nucleic acid molecule into acell to produce a transformed cell; and growing the transformed cell toproduce at least one protein.
 18. The method of claim 17 wherein saidfirst nucleic acid molecule contains a portion encoding the leadersequence for said non-proteolytic analog.
 19. The method of claim 18wherein said non-proteolytic analog of Hin47 protein is a mutation ofnatural Hin47 protein in which at least one amino acid selected from thegroup consisting of amino acids 91, 121 and 195 to 201 of natural Hin47protein has been deleted or replaced by a different amino acid.
 20. Themethod of claim 19 wherein histidine 91 is replaced by alanine.
 21. Themethod of claim 20 wherein said at least one additional nucleic acidmolecule encodes a Hia or Hsf protein of a strain of Haemophilusinfluenzae.
 22. The method of claim 21 wherein said at least oneadditional nucleic acid molecule encodes a Hia protein which isN-terminally truncated.
 23. The method of claim 22 wherein saidN-terminal truncation is S44 or V38.
 24. The method of claim 21 whereinsaid vector is a plasmid vector having the identifying characteristicsof plasmid JB-3145-1 as shown in FIG.
 10. 25. The method of claim 20wherein said at least one additional nucleic acid molecule encodes aPsaA protein of a strain of Streptococcus pneumoniae.
 26. The method ofclaim 17 wherein said first nucleic acid molecule encodes the natureform of the non-proteolytic analog.
 27. The method of claim 26 whereinsaid non-proteolytic analog of Hin47 protein is a mutation of naturalHin47 protein in which at least one amino acid selected from the groupconsisting of amino acids 91, 121 and 195 to 201 of natural Hin47protein has been deleted or replaced by a different amino acid.
 28. Themethod of claim 27 wherein histidine 91 is replaced by alanine.
 29. Themethod of claim 28 wherein said at least one additional nucleic acidmolecule encodes a Hia or Hsf protein of a strain of Haemophilusinfluenzae.
 30. The method of claim 29 wherein said at least oneadditional nucleic acid molecule encodes a Hia protein which isN-terminally truncated.
 31. The method of claim 30 wherein saidN-terminal truncation is S44 or V38.
 32. The method of claim 31 whereinsaid vector is a plasmid vector having the identifying characteristicsof plasmid DS-2342-2-2 as seen in FIG.
 5. 33. The method of claim 31wherein said vector is a plasmid vector having the identifyingcharacteristics of plasmid JB-3134-1-1 as seen in FIG.
 9. 34. The methodof claim 28 wherein said at least one additional nucleic acid moleculeencodes a PsaA protein of a strain of Streptococcus pneumoniae.
 35. Themethod of claim 34 wherein said vector is a plasmid vector having theidentifying characteristics of plasmid JB-3073R-1 as seen in FIG. 12.36. The method of claim 34 wherein said vector is a plasmid vectorhaving the identifying characteristics of plasmid JB-3090-1 or JB-3090-7as seen in FIG.
 13. 37. An expression vector, comprising: a nucleic acidmolecule encoding a high molecular weight protein of a non-typeablestrain of Haemophilus selected from the group consisting of hmwB andhmwC, and a promoter operatively connected to said nucleic acid moleculeto direct expression of said high molecular weight protein in a hostcell.
 38. The vector of claim 37 which is a plasmid vector having theidentifying characteristics of plasmid IN-137-1-16 shown in FIG. 18A.39. The vector of claim 37 which is a plasmid vector having theidentifying characteristics of pT7 hmwC shown in FIG. 19A.
 40. Anexpression vector for expression of a recombinant protein in a hostcell, comprising: a nucleic acid molecule encoding a high molecularweight (HMW) protein of a non-typeable strain of Haemophilus selectedfrom the group consisting of hmwBC, hmwB and hmwC, at least oneadditional nucleic acid molecule encoding the recombinant protein, andat least one regulatory element operatively connected to said firstnucleic acid molecule and said at least one additional nucleic acidmolecule to effect expression of at least said recombinant protein inthe host cell.
 41. The expression vector of claim 40 wherein said atleast one additional nucleic acid molecule is inserted into a plasmidhaving the identifying characteristics of plasmid IN-52-1-13 as shown inFIG. 17A and under control of said at least one regulatory element. 42.The expression vector of claim 40 wherein said at least one additionalnucleic acid molecule is inserted into a plasmid having the identifyingcharacteristics of plasmid IN-137-1-16 shown in FIG. 18A and undercontrol of said at least one regulatory element.
 43. The expressionvector of claim 40 wherein said at least one additional nucleic acidmolecule is inserted into a plasmid having the identifyingcharacteristics of plasmid pT7 hmwC shown in FIG. 19A and under controlof said at least one regulatory element.